Journal: bioRxiv

Article Title: CLCC1 promotes membrane fusion during herpesvirus nuclear egress

doi: 10.1101/2024.09.23.614151

Figure Lengend Snippet: a) The flow-cytometry-based nuclear egress assay separates HSV-1 infected HeLa cells with vs. without nuclear egress based on two fluorescent signals: tdTomato (red, HSV-1 infection) and Alexa-488 (green, presence of cytoplasmic capsids). HeLa cells infected with HSV-1 encoding tdTomato, were partially permeabilized 24 hpi and stained with an anti-capsid mAb and an Alexa488-conjugated secondary mAb. Cells in the tdTomato+/Alexa488+ quadrant (Q2) are infected and have cytoplasmic capsids, indicating nuclear egress. Cells in the tdTomato+/Alexa488-quadrant (Q1) are infected but do not have cytoplasmic capsids, indicating no nuclear egress. Left: ∼90% cells infected with WT HSV-1 are tdTomato+/Alexa488+. Right: only ∼6% cells infected with HSV-1 ΔUL34 virus, which has a defect in nuclear egress, are tdTomato+/Alexa488+. b) Schematic of the genome-wide CRISPR screen. HeLa-Cas9 cells were transduced with Gattinara library lentivirus, containing ∼40,000 sgRNAs, with 2 sgRNAs/gene, and after selection with puromycin, were infected with HSV-1 encoding tdTomato. 24 hpi, partially permeabilized cells were stained with a capsid-specific antibody and sorted by flow cytometry. c) Volcano plot of the screen results. Each dot represents a specific gene. The x-axis shows the fold change (FC) of sgRNAs, plotted as log(FC). Genes with log(FC) values >0 or <0 are candidate positive or negative regulators, respectively. The y-axis shows the significance score plotted as -log(p-value). The dotted line at y = 3 is the threshold for p-value < 0.001, indicating high confidence candidates. The dashed line at y = 1.3 is the threshold for p-value < 0.05. Red: top hit, CLCC1. Green: genes known to contribute to HSV-1 nuclear egress. High-confidence hit, EMD, is labelled. Gray: control sgRNAs (targeted, non-site, or intergenic sites).

Article Snippet: Lentiviral sgRNA Gattinara library (CP0073) targeting the whole genome (19993 target genes, 40964 sgRNAs) was provided by the Genetic Perturbation Platform group at the Broad Institute.

Techniques: Flow Cytometry, Infection, Staining, Virus, Genome Wide, CRISPR, Transduction, Selection, Control

Journal: bioRxiv

Article Title: CLCC1 promotes membrane fusion during herpesvirus nuclear egress

doi: 10.1101/2024.09.23.614151

Figure Lengend Snippet: A) Schematic representation of the locations of CLCC1 mutations and their functions (if known). Structural elements and domains were assigned as in and colored as follows: TM1/TM2/TM3 (light blue), FD (light pink), and AH (light orange). S-S = predicted disulfide bond (purple). Approximate locations of mutated residues are shown as dots. b) A ribbon diagram of an AlphaFold3 model of human CLCC1. Structural elements and domains are colored as in (a) and labelled. Mutated residues are shown in sphere representation and colored as in a) . Residues 365-539 were removed, for clarity. c, d) Several CLCC1 mutants rescue a defect in nuclear egress due to CLCC1 depletion whereas others do not, as measured by the flow cytometry nuclear egress assay. Single-clone CLCC1-KO (cko6_1) (c) or control Int_4 cell line (d) were either mock-transduced (Mock) or transduced with lentiviral constructs encoding WT CLCC1 or mutants D152R/D153R, E175R/D176R, D181R, D25E/D181R, S263R, W267R, D277R, K298E in the CLCC1-CR background. Bulk pools were infected with WT HSV-1 at an MOI of 5. Nuclear egress was measured at 24 hpi and normalized to the Int_4 mock. Each experiment had at least two biological replicates, each containing two technical replicates. Each data point represents a biological replicate. Bars represent mean values, and the error bars represent SEM. P < 0.01 = **, P < 0.05 = *. Significance was calculated using one-way ANOVA, with multiple comparisons. The color scheme is as in (a) and (b).

Article Snippet: Lentiviral sgRNA Gattinara library (CP0073) targeting the whole genome (19993 target genes, 40964 sgRNAs) was provided by the Genetic Perturbation Platform group at the Broad Institute.

Techniques: Flow Cytometry, Control, Transduction, Construct, Infection